Mitoregulin self-associates to form likely homo-oligomeric pore-like complexes

We and others previously found that a misannotated long noncoding RNA encodes for a conserved mitochondrial transmembrane microprotein named Mitoregulin (Mtln). Beyond an established role for Mtln in lipid metabolism, Mtln has been shown to broadly influence mitochondria, boosting respiratory efficiency and Ca²⁺ retention capacity, while lowering ROS, yet the underlying mechanisms remain unresolved. Prior studies have identified possible Mtln protein interaction partners; however, a lack of consensus persists, and no claims have been made about Mtln's structure. We noted two key published observations that seemingly remained overlooked: 1) endogenous Mtln co-immunoprecipitates with epitope-tagged Mtln at high efficiency, and 2) Mtln primarily appears to exist in a ∼66 kDa complex. To investigate if Mtln may self-oligomerize into higher-order complexes, we performed co-immunoprecipitation, computational modeling, and native gel assessments of Mtln-containing complexes in cells and tissues and tested whether synthetic Mtln protein itself forms oligomeric complexes. Our combined results provide strong support that Mtln self-associates and likely forms a hexameric pore-like structure.